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Journal of Virology, October 2001, p. 10005-10013, Vol. 75, No. 20
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.20.10005-10013.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Differentiation-Dependent Chromatin Rearrangement
Coincides with Activation of Human Papillomavirus Type 31 Late
Gene Expression
Loren del Mar
Peña and
Laimonis A.
Laimins*
Department of Microbiology-Immunology,
Northwestern University Medical School, Chicago, Illinois 60611
Received 29 May 2001/Accepted 13 July 2001
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ABSTRACT |
The life cycle of human papillomaviruses (HPVs) is tightly linked
to the differentiation status of the host cell. While early genes are
expressed during the initial stages of viral infection, late gene
expression occurs in the suprabasal layers of the cervical epithelium.
Late genes encode E1^E4, a cytosolic
protein, and capsid proteins L1 and L2. We have mapped over 30 initiation sites for late transcripts and show that the transcripts
initiate in a 200-nucleotide region within the E7 open reading frame.
The mechanisms regulating the activation of late gene expression, however, are not yet understood. DNase I hypersensitivity analysis of
HPV-31 chromatin in cell lines that maintain viral genomes extrachromosomally indicates that a major shift in nuclease digestion occurs upon differentiation. In undifferentiated cells, hypersensitive regions exist in the upstream regulatory region proximal to the E6 open
reading frame. Upon differentiation, a region between nucleotides 659 and 811 in the E7 open reading frame becomes accessible to DNase I. These results indicate that the late transcript initiation region
becomes accessible to transcription factor binding upon differentiation. Several complexes mediate chromatin rearrangement, and
we tested whether histone acetylation was sufficient for late transcript activation. Treatment with the histone deacetylase inhibitor
trichostatin A was found to be insufficient to activate late gene
expression in undifferentiated cells. However, it did activate
expression of early transcripts. These results suggest that chromatin
remodeling around the late promoter occurs upon epithelial
differentiation and that mechanisms in addition to histone
deacetylation contribute to activation of late gene expression.
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TEXT |
Papillomaviruses (PVs) are a family
of small DNA viruses that infect epithelial tissue at various
anatomical locations. Over 85 PV types that are specific for humans
(HPVs) have been identified, and they show a high degree of sequence
and functional conservation. A subset of HPVs infect the genital
epithelia and are broadly divided into two categories: low-risk viruses
that cause benign hyperproliferative lesions, and high-risk types that
can potentially progress to neoplastic transformation
(23).
High-risk HPV types include HPV-16, -18, -31, -33, -45, and -54 (23, 35). During the initial stages of HPV infection, viral DNA is established as episomes in the basal layer of stratified epithelia, with a copy number of 50 to 100 copies per cell. Early transcripts are expressed from a promoter upstream of E6 and, in
HPV-31, initiate at approximately nucleotide 97 (p97) (Fig. 1A) (25). Similar promoters
have been identified in HPV-16 and -18 (4, 7). Early
transcripts are polycistronic messages that encode a number of viral
proteins, including E1, E2, E6, and E7 (25, 47). The early
promoter is regulated by sequences within a noncoding upstream
regulatory region (URR) containing the origin of viral replication as
well as binding sites for viral and cellular proteins (23, 24,
28, 34, 54). Copy number and early gene expression are tightly
regulated at this initial stage by viral proteins E1 and E2 (13,
14, 17, 36). As basal cells divide and daughter cells migrate
from basal strata, they begin to differentiate. As differentiation
occurs, two viral processes take place: viral genome amplification and
late gene induction (6, 18). Late genes consist of
polycistronic messages that include E1^E4,
a cytoplasmic protein thought to facilitate viral egress from infected
cells, as well as L1 and L2, the two viral capsid proteins (Fig. 1B)
(15, 21, 25, 26, 40, 43, 44). Keratinocyte differentiation
is therefore required for a productive viral infection.
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FIG. 1.
(A) Linear representation of the HPV-31 genome. Open
reading frames are indicated underneath the map. PE refers
to the early promoter (p97); PL refers to the late
promoter. PolyA indicates polyadenylation sites. (B) Cartoon depicting
late HPV-31 transcripts. PL designates the late promoter.
Splice sites are indicated by the numbers below each transcript. Open
reading frames are specified to the right of each message.
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Past work from Meyers et al. used organotypic raft culture to mimic the
complete HPV-31 life cycle in vitro using the CIN 612 cell line, which
was established from a cervical biopsy that maintained episomal copies
of HPV-31b (38). The raft system successfully induced
epithelial differentiation, viral genome amplification, induction of
late genes, and assembly of infectious virions. Additional studies
identified late transcripts that appeared upon differentiation of CIN
612 cells and initiated in a region around nucleotide 742 (Fig. 1)
(25, 26). Differentiation-induced transcripts also appear
in HPV-16 and HPV-6; furthermore, these transcripts initiate in a
similar region of the E7 open reading frame (ORF) (20, 22, 29,
39). While some of the initiation sites of the late promoter
have been mapped, a detailed analysis of the extent of these initiation
sites is incomplete.
The mechanisms that mediate the switch from early to late promoter
usage have not been elucidated, and the role of chromatin in this
regulation has not been established. Nuclear DNA is condensed into
chromatin and, in this state, is generally inaccessible to the
transcriptional machinery (59). In contrast,
transcriptionally active regions of DNA are readily accessible to
transcription factors. The basic unit of chromatin is the nucleosome, a
complex of approximately 200 nucleotides tightly wound around a core
octamer of histones H2A, H2B, H3, and H4 and one linker histone.
Nucleosomes are assembled into higher-order structures of increased
compaction. It is generally agreed that chromatin remodeling must occur
for the transcriptional machinery to gain access to DNA
(59). Two cellular processes facilitate gene expression
via chromatin modification: ATP-dependent chromatin remodeling and
histone acetylation (8, 30, 33). ATP-dependent remodeling
is thought to be a catalytic process that shifts the thermodynamic
equilibrium between different nucleosomal conformations to favor the
relaxed state. A second process is mediated by histone
acetyltransferases (HATs) and covalently modifies histones. HATs add
acetyl moieties to lysine residues in histone tails; as a result,
acetylated histones are unable to bind DNA tightly. The process can be
reversed by histone deacetylases.
While it has been observed that HPV late transcript expression requires
epithelial differentiation, the process by which late gene expression
is activated remains unknown. It is possible that differentiated cells
express cellular factors that are required for late gene expression or
that viral genome amplification results in viral chromatin
rearrangement to make it more accessible to the transcriptional
machinery. In this study, we have mapped in detail the initiation sites
for late transcripts and examined chromatin remodeling around the
HPV-31 late promoter at different stages of the viral life cycle.
Late transcripts initiate at various sites within the E7 ORF.
The HPV-31 life cycle includes a temporal pattern of expression of
early and late genes. Late gene expression requires epithelial differentiation, and late transcripts have been observed to initiate at
heterogeneous sites (20, 22, 29, 39). Previous studies induced differentiation by means of organotypic raft cultures, which
provided a mix of both undifferentiated and differentiated cells. We
have recently determined that differentiation induced by suspension in
1.5% methylcellulose for 24 h, in contrast to raft culture,
resulted in a relatively uniform degree of differentiation in all cells
and induced viral DNA amplification and late transcript expression
(45, 46).
An RNase protection assay is shown in Fig.
2A, in which we compared initiation site
usage between early and late genes. RNA
samples from an
undifferentiated population of CIN 612 cells (lane
4) and from CIN 612 cells differentiated in methylcellulose for
24 h (lane 5) were
hybridized to a probe (pRP-p742) that detects
early and late
transcripts (
51). Two bands representing early
transcripts
are seen in both differentiated and undifferentiated
cells. These bands
correspond to spliced and unspliced messages
initiated at the early
promoter. In contrast, late transcripts
are induced upon
differentiation and consist of several bands
corresponding to a number
of initiation sites (compare lane 4
with lane 5). The pattern of
initiation among unspliced and spliced
late messages appears to differ;
however, we have not determined
whether splicing events affect
initiation site usage among late
transcripts. Previous work from Hummel
et al. and Ozbun and Meyers
indicated that a number of late messages
initiate at nucleotides
other than nucleotide 742 (
25,
26,
40,
41). We therefore
performed a more extensive analysis to map the
differentiation-dependent
transcripts by RNase protection assay on RNA
isolated from the
CIN 612 cell line.
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FIG. 2.
RNase protection assays. Total RNA was isolated from
monolayer or differentiated cells using the Trizol reagent (Invitrogen,
Carlsbad, Calif.) per the manufacturer's instructions. Antisense
riboprobes labeled with 32P were synthesized using the
Riboprobe transcription system from Promega. Probes were purified
following gel electrophoresis in 6% acrylamide. RNase protection assay
was performed as described by Stubenrauch et al. (50),
with the following exceptions: 15 µg of RNA was used for each
hybridization to 1.5 × 105 cpm of probe, and the RNA
was digested with 12-µg/ml RNase A and 60-U/ml RNase T1
(Roche Molecular Biochemicals, Indianapolis, Ind.). Labeled 100-bp
ladder (Invitrogen) and a DNA sequencing ladder were run with samples
as size markers. Lanes contain hybridization samples as follows: 1, undigested probe; 2, no RNA; 3, yeast tRNA; 4, RNA from monolayer CIN
612 cells; 5, RNA from CIN 612 cells induced to differentiate in
methylcellulose for 24 h. (A) The pRP-p742 probe, which detects
both early and late messages, has been described previously
(50). The probe is illustrated by a stippled rectangle
below the autoradiograph. Corresponding open reading frames are
specified under the probe. PL designates the late promoter,
while SD877 represents a splice donor site at nucleotide 877. Early and
late transcripts are marked to the right of the bands. (B to D) RNase
protection assay for mapping of late transcripts. All probes are
specified below each autoradiograph. The probe used in panel B spans
nucleotides 600 to 850; the probe in panel C spans nucleotides 600 to
756; the probe in panel D includes nucleotides 750 to 850. The numbers
to the right of each band indicate the nucleotide location of each
initiation site. The slowest-migrating band in lanes 4 and 5 in each
panel corresponds to the fully protected probe.
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We mapped transcript initiation sites for the late promoter in the E7
ORF using five overlapping probes that spanned approximately
300 nucleotides within the E7 ORF. To ensure that our studies
identified
real start sites, we required that the putative sites
be detected in
analysis with at least two overlapping probes.
None of these probes
spanned a known splice donor or acceptor
site. The transcripts were
mapped by size comparison with a DNA
sequencing ladder, and three
representative experiments are shown
in Fig.
2B to
2D. Because RNA has
a lower mobility in denaturing
polyacrylamide gels than DNA of the same
size, transcript initiation
sites were mapped within a 5-nucleotide
margin of
error.
We observed a set of protected bands in differentiated cells that
corresponded to approximately 35 different initiation sites
within the
E7 ORF. Three representative RNase protection assays,
which use three
different probes that detect 18 initiation sites,
are shown on Fig.
2B
to
2D. The frequency of initiation site usage
varied among transcripts,
as judged by band intensity on RNase
protection assay. Interestingly,
several protected bands were
observed to initiate in the E7 ORF in
undifferentiated cells as
well as in differentiated cells. These
transcripts therefore appear
to be expressed constitutively throughout
the life cycle of the
virus (Fig.
2B and
2C). An example of these
messages included
a transcript initiating at nucleotide 706 that is
expressed in
monolayer CIN 612 cells and is upregulated in cells that
have
undergone differentiation (Fig.
2B). The region of
differentiation-dependent
transcript initiation that we identified
spans approximately 200
nucleotides within the E7 ORF. Figure
3 summarizes initiation
site usage within
the E7 ORF. The 35 transcripts that we mapped
include initiation sites
at nucleotides 737, 742, 750, and 767,
consistent with previous reports
mapping some of these sites (
40,
41).
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FIG. 3.
Partial linear map of HPV-31 with corresponding open
reading frames at the bottom. The portion of the E7 ORF from
nucleotides 600 to 850 has been magnified to mark with arrows the
initiation sites that were identified in this study. Initiation sites
that show a high frequency of usage are underlined. Transcripts that
are constitutively expressed are indicated by asterisks. Sequences in
dashed boxes are initiator elements, and the CCAAT box is indicated by
a dashed oval.
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A change in chromatin architecture around the late initiation
region coincides with epithelial differentiation.
The mechanism by
which late genes are induced remains unclear. PV genomes are associated
with histone proteins (16), and chromatin remodeling has
been shown to affect gene expression (5, 9, 55, 58, 60,
61). We therefore examined the state of chromatin throughout the
life cycle of HPV-31 in order to observe any changes in the
architecture of viral chromatin. The DNase I hypersensitivity assay was
used, as it allows us to observe patterns of permeability to nuclease
digestion throughout the genome at different stages of the viral life
cycle. This assay is based on the fact that nucleosomal arrays are
resistant to nuclease cleavage, while regions associated with
nonhistone proteins are sensitive to DNase I (10).
Furthermore, the pattern of DNase I sensitivity has been shown to
correlate with the binding of transcriptional regulators to
hypersensitive sites.
For this study, we isolated nuclei from undifferentiated and
methylcellulose-differentiated populations of CIN 612 cells by
sucrose
gradient centrifugation. Following isolation, the nuclei
were lightly
treated with various concentrations of DNase I. DNA
extracts from
treated nuclei were prepared and digested with
BlpI
and
AlwNI, each of which cleaves the HPV-31 genome at a single
site. Digestion with these two enzymes yields a subgenomic restriction
fragment from nucleotides 6269 to 1097, which spans the 3' half
of the
L1 ORF to the 5' half of the E1 ORF (Fig.
4A). Because
the nuclei were lightly
treated with DNase I, a portion of the
HPV-31 molecules escaped
nuclease digestion. This full-length
BlpI-
AlwNI
restriction fragment ran as a 2.7-kb "parental" band
(Fig.
4A). The
digested DNA was examined by Southern analysis
using a subgenomic probe
positioned at the 3' end of the
BlpI-
AlwNI
restriction fragment. The sites of nuclease digestion were mapped
by
their distance from the 3' end of the probe.
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FIG. 4.
DNase I hypersensitivity analysis of HPV-31. (A)
Schematic representation of the parental fragment, probe, and
corresponding regions in HPV-31. P97, early promoter. URR and open
reading frames are indicated by boxes. Restriction sites are indicated
by italics: BlpI; BanII;
A, AlwNI; S,
SpeI; H, HpaI. (B)
Autoradiograph corresponding to a DNase I hypersensitivity assay. Lanes
1 to 5, nuclei from undifferentiated CIN 612 cells. Lanes 6 to 10, nuclei from CIN 612 cells that were induced to differentiate in
semisolid medium for 24 h. Nuclei were treated with a titration of
DNase I corresponding to 10, 5, 2.5, 1.25, and 0 U/µl. Genomic DNA
was digested with BlpI and AlwNI
restriction endonucleases to yield a 2.7-kb parental fragment, and
areas of hypersensitivity were observed by Southern blot analysis with
a probe corresponding to the BanII-AlwNI
fragment of HPV-31. Subgenomic marker fragments used for mapping areas
of nuclease digestion are indicated at the left. Regions of the HPV-31
genome that show hypersensitivity are indicated to the right of the
autoradiograph.
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Several species of higher mobility were detected in nuclei treated with
DNase I. An area of hypersensitivity was observed
in the vicinity of
the
SpeI-
AlwNI marker in undifferentiated CIN
612 cells, between nucleotides 7557 and 215 (Fig.
4B, lanes 1
to 4). This
region corresponds to sequences within the URR and
proximal to the
early promoter. This pattern of hypersensitivity
is absent in nuclei
that were not treated with DNase I (lane 5),
and its intensity
increases proportionally to the concentration
of DNase I that was used.
The location of these DNase I-hypersensitive
sites is in accordance
with published studies of HPV-31 promoter
usage in undifferentiated
cells.
We detected a major shift in the pattern of hypersensitivity in cells
that had undergone differentiation in methylcellulose.
An area of
sensitivity mapping close to marker fragment
BanII-
AlwNI
appeared in differentiated CIN
612 cells (Fig.
4B, lanes 6 to
9). This area of nuclease digestion
occurs within the region between
nucleotides 659 and 811. The observed
digestion pattern was absent
from untreated nuclei and was not seen in
nuclei isolated from
undifferentiated cells at similar concentrations
of DNase I. The
area of hypersensitivity maps within the E7 ORF and
corresponds
to the region where we mapped a number of initiation sites
for
differentiation-dependent transcripts. We have observed the same
pattern of hypersensitivity in at least three independent experiments
using CIN 612 cells at different passage. The pattern of nuclease
digestion suggests that DNA accessibility around the E7 ORF changes
with differentiation so that the DNA becomes associated with nonhistone
proteins, which could potentially recruit the transcriptional
machinery
to the region. We also observed a decrease in accessibility
to DNase I
in the proximity of the early promoter upon differentiation
(Fig.
4B,
lanes 6 to
9).
Histone hyperacetylation is insufficient to activate late
transcript expression.
Because histone acetylation is one
mechanism that mediates chromatin remodeling, we tested whether it
could induce an altered chromatin configuration similar to that seen
upon epithelial differentiation. If changes in histone acetylation were
solely responsible for altered DNase I hypersensitivity, we would
expect that treatment of undifferentiated cells with an inhibitor of
histone deacetylases would lead to the same pattern of sensitivity seen
in differentiated cells. Undifferentiated monolayer cultures of CIN 612 cells were therefore treated with various concentrations of
trichostatin A (TSA), a noncompetitive inhibitor of histone
deacetylases (62, 63), and harvested after 24 h. We
then performed DNase I hypersensitivity analysis and observed that TSA
treatment of monolayer cells did not induce a significant degree of
hypersensitivity in the E7 ORF (data not shown).
It is possible that any chromatin alterations induced by histone
hyperacetylation in the TSA-treated cells occur in only a
subset of
cells or are too subtle to be detected by Southern blotting.
We
therefore performed RNase protection assays as a functional
test for a
relationship between histone hyperacetylation and late
gene expression.
Antisense riboprobe pRP-p742, which detects both
early and late
transcripts, was hybridized to RNA harvested after
treatment with TSA,
and an RNase protection assay was performed
(Fig.
5A) (
51). As shown in Fig.
5A, histone hyperacetylation
does not induce late transcript activation
in monolayer cells
(lanes 5 to 7). This is in contrast to the strong
activation of
late gene expression in cells that were induced to
differentiate
in methylcellulose (lane 8). These results suggest that
late gene
expression is linked to a change that occurs upon epithelial
differentiation
and that histone acetylation alone is not responsible
for late
gene induction.
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FIG. 5.
(A) RNase protection assay. Probe pRP-p742 was used to
examine whether histone hyperacetylation induced late gene expression
after treatment of undifferentiated CIN 612 cells with TSA
(Sigma-Aldrich, St. Louis, Mo.). TSA was dissolved in ethanol and
diluted directly into culture media to a final concentration of 100, 300, or 900 nM. The same volume of ethanol was added to the untreated
samples. Monolayer cells were treated for 24 h and used directly
to isolate RNA or induced to differentiate in semisolid medium for
24 h. Lane 1, undigested probe; lane 2, no RNA; lane 3, yeast
tRNA; lane 4, untreated monolayer CIN 612 cells; lanes 5 to 7, undifferentiated CIN 612 cells treated with TSA at 100 nM (lane 5), 300 nM (lane 6), or 900 nM (lane 7). Lane 8 shows the pattern of late gene
expression induced following suspension in methylcellulose for 24 h in the absence of TSA. Transcripts are labeled to the right of bands.
The low levels of late transcript initiation in undifferentiated cells
are due to a small percentage of cells that differentiate spontaneously
(43). (B) Southern blot analysis for episome copy number
after treatment with TSA. The Southern blot assay was performed as
previously described (24). Briefly, 10 µg of sheared
genomic DNA was digested with BglII (New England
Biolabs), an enzyme that does not cut the HPV-31 genome, or with
HpaI (New England Biolabs), a single cutter in HPV-31.
The digests were separated in 0.8% agarose, transferred to a ZetaProbe
GT membrane (Bio-Rad), and hybridized to an HPV-31 genomic probe at
42°C. Lanes: 1, untreated monolayer CIN 612 cells; 2 to 4, monolayer
CIN 612 cells treated with 100, 300, and 900 nM TSA, respectively; 5, untreated CIN 612 cells that were induced to differentiate in semisolid
medium for 24 h. All bands were quantified by PhosphorImager
analysis.
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Interestingly, our studies suggest that histone acetylation may play a
role in regulation of early transcript expression.
PhosphorImager
analysis indicates that early transcript expression
is upregulated
approximately threefold in samples treated with
900 nM TSA (data not
shown). This number is an average of two
different experiments. Because
histone acetylation also plays
a role in DNA synthesis (
1,
12,
32), we decided to investigate
whether the increase in early
transcripts was correlated with
an increase in viral template number
after TSA treatment. Undifferentiated
monolayer cultures of CIN 612 cells were treated with TSA for
24 h, DNA extracts were prepared,
and viral copy number was determined
by Southern blot analysis.
PhosphorImager analysis revealed that
episomal DNA was increased in
undifferentiated samples treated
with 900 nM TSA by 1.8-fold relative
to the untreated cells (Fig.
5B). This increase in copy number after
TSA treatment may partly
account for increased early transcripts in
undifferentiated
cells.
Western blot analysis was performed to detect the extent of histone
acetylation after treatment with TSA. Undifferentiated
CIN 612 cells
were treated with TSA for 24 h, and protein extracts
were prepared
for Western blotting. We observed a dose-dependent
increase in the
levels of acetylated H4 following TSA treatment
(data not shown). These
results indicate that, while TSA treatment
does induce histone
hyperacetylation, this effect is insufficient
to activate late gene
expression.
The HPV life cycle is tightly linked to the differentiation status of
the host cell. Two sets of viral genes follow a temporal
pattern of
expression: early genes are expressed throughout the
life cycle, while
late genes require epithelial differentiation
for expression. In this
study, we have performed RNase protection
assays to map HPV-31
transcripts that initiate at various sequences
within the E7 ORF.
Previous studies mapped transcript initiation
sites at nucleotides 737, 742, 750, and 767. In this study, we
mapped approximately 35 additional
start sites that span 200 nucleotides
within the E7 ORF. These 5'
termini include start sites at nucleotides
626, 642, 651, 680, 733, and
751. In addition, we observed that
a number of transcripts that
initiate in the E7 ORF are expressed
constitutively throughout the life
cycle. For example, a transcript
initiating at nucleotide 706 was
expressed in monolayer cells,
although at lower levels than observed
when cells underwent differentiation.
These constitutive transcripts
could provide low-level expression
of the replication proteins E1 and
E2 (
31). The promiscuous
initiation site usage that we
observed among HPV-31 late transcripts
is similar to that of other DNA
tumor viruses such as simian virus
40 (SV40). In the case of SV40,
approximately two dozen initiation
sites for late transcripts have been
identified, and they span
a 300-nucleotide region of the genome
(
49).
No consensus TATA box (TATAAA) is present in the E7 ORF of HPV-31,
HPV-6, HPV-16, or bovine PV type 1 (
3,
20,
22,
29,
39). In
the case of most TATA-less promoters, an initiator element
overlaps the
initiation site and performs an analogous function
to the TATA box: it
directs assembly of the basal transcriptional
machinery at the correct
initiation site (
10). Sequence analysis
indicates that
initiator elements may be present at nucleotides
653 to 659, 736 to
742, and 747 to 753 of HPV-31. Some of the
start sites that we mapped
overlap these putative elements. The
rest of the transcripts, however,
lack both a TATA box and initiator
element. Transcription from
TATA-less and initiatorless promoters
appears to be mediated by
transcription factors, such as Sp1,
that presumably direct the
transcriptional machinery to particular
initiation sites. Initiation
from this type of promoter has been
shown to occur in a promiscuous
manner over hundreds of nucleotides
throughout the promoter
(
48).
Sequence analysis of the E7 ORF using the TRANSFAC database
(
57) reveals that a number of potential transcription
factor-binding
sites occur in this region. In preliminary studies, we
observed
bandshifts in electrophoretic mobility shift assays using
probes
that spanned the E7 ORF and identified in vitro binding of
Oct-1,
SOX5, and SRY (L. Peña, unpublished data). It is therefore
possible
that these binding sites function as transcriptional
cis elements
to direct expression of late transcripts. It is
also possible
that sequences outside the E7 ORF, such as the URR, play
a role
in late gene
expression.
One model for late gene activation involves transcription factors that
are expressed exclusively upon differentiation and
direct late
transcript initiation within the E7 ORF. Alternatively,
the viral
replication that occurs at suprabasal strata may play
an important role
in activating late gene expression through one
of two mechanisms. One
possibility is that viral DNA replication
leads to increased template
number, so that low levels of constitutive
late transcript expression
can now be detected. A second possibility
is that viral genome
amplification titrates a repressor of late
gene expression in a manner
similar to that in SV40 (
56). Both
models require episomal
templates for activation of late gene
expression and are consistent
with two previous observations.
First, Frattini et al. showed that
HPV-31 must exist as an episome
for late gene expression to be
activated (
19). Second, CIN 612
cells that spontaneously
amplify viral DNA in monolayer cultures
also express late genes
(
43).
It is generally accepted that the state of chromatin can have a major
impact on gene expression. DNA that is densely packaged
into
nucleosomes is inaccessible to the transcriptional machinery,
and
expression of a particular gene is therefore contingent on
modification
of the chromatin in the region. In our study, we
used the DNase I
hypersensitivity assay to observe the configuration
of viral chromatin
throughout the life cycle of HPV-31 and observed
a major shift in
nuclease hypersensitivity upon epithelial differentiation.
Differentiated cells show increased permeability to DNase I digestion
in the vicinity of nucleotides 659 and 811. This region is located
within the E7 ORF and encompasses the late transcript initiation
sites
that we mapped by RNase protection assay. The observed shift
in
hypersensitivity suggests that transcription factor binding
occurs in
the E7 ORF upon epithelial
differentiation.
SV40 gene expression is a prominent example of the role of chromatin
remodeling in transcription. A series of studies indicate
that
nucleosome positioning along the SV40 origin of replication,
the early
promoter, and the late promoter affects the activity
of these elements
(
2,
27,
42,
53). Cereghini et al. have
observed that a
DNase I-hypersensitive site along the late transcript
initiation region
appears during the switch to late gene expression
(
11).
Chromatin remodeling therefore leads to increased accessibility
to the
SV40 late promoter and facilitates late gene
expression.
We have identified a change in the in vivo chromatin structure of
HPV-31 in the vicinity of the early promoter and the late
transcript
initiation region. Our data suggest that a change in
differentiated
cells coincides with chromatin remodeling in the
E7 ORF, which is in
turn associated with late gene expression.
Studies by Stünkel and
Bernard provide evidence that chromatin
rearrangement plays a role in
HPV gene expression (
52). In vivo
studies of CaSki cells,
which carry approximately 500 copies of
integrated HPV-16, indicate
that nucleosome positioning in the
URR occurs over the viral enhancer,
the origin of viral replication,
and the early promoter. The presence
of a nucleosome over the
HPV-16 early promoter has a repressive effect
on promoter activity.
In vitro studies also showed that a nucleosome
assembles over
the early promoter of HPV-18 at approximately the same
site as
in HPV-16. These results suggest that viral chromatin
organization
may be dictated by the DNA sequence and that it regulates
viral
gene
expression.
Several cellular complexes remodel chromatin so that the DNA template
can be accessed for transcription. These include histone
acetylation
and ATP-dependent remodeling complexes. Although they
differ in
function, both complexes increase DNA accessibility
to the
transcriptional machinery. Histone acetylation decreases
the affinity
of histone protein binding to DNA and leads to transcriptional
activation of a number of promoters. Our analysis of TSA-treated
CIN
612 cells indicates that histone acetylation is not sufficient
to
activate late gene expression in undifferentiated cells. It
is possible
that the altered architecture in differentiated cells
is induced by
another chromatin remodeling complex, such as SWI/SNF.
Interestingly,
human SNF5 was recently reported to interact with
the viral protein E1
and participate in viral DNA replication
(
37). Whether
this interaction plays a role in late promoter
activation remains to be
seen.
Our studies suggest that histone acetylation may play a role in HPV
early gene expression from episomal templates. Zhao et
al. have also
reported increased transcription from the URR-E6
promoter of HPV-11
after TSA treatment (
64). In addition, we
observed a
1.8-fold increase in episome copy number in undifferentiated
cells
treated with TSA. In contrast, differentiation induces,
on average, a
threefold increase in viral copy number. The increase
in copy number
that was observed in TSA-treated cells may therefore
be insufficient to
activate late gene expression. It is also possible
that, while copy
number may play an integral role in late gene
induction, other
processes may also be at work. Additional studies
examining the
sequences that regulate induction of the late promoter
will be required
to further understand the mechanisms that regulate
differentiation-dependent viral gene
expression.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from the National Cancer
Institute to L. Peña (1F31CA80673-01) and to L. Laimins (CA59655).
We thank members of the Laimins laboratory for technical advice and A. Aiyar and K. Rundell for helpful comments on the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology-Immunology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Phone: (312) 503-0648. Fax: (312)
503-0649. E-mail: l-laimins{at}northwestern.edu.
| |
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Journal of Virology, October 2001, p. 10005-10013, Vol. 75, No. 20
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.20.10005-10013.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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